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Changes in cyclin dependent kinase expression and activity accompanying lens fiber cell differentiation
C Y Gao1, A M Rampalli, H C Cai
1Laboratory of Molecular and Developmental Biology, NEI/NIH, Bethesda, MD, 20892-2730, USA.
Abstract:
Previous studies from this laboratory have shown that differentiating lens fiber cells contain two active cyclin dependent kinases (Cdks), Cdk1 and Cdk5. The present study was undertaken to explore the expression and regulation of six additional members of the Cdk family (Cdk2, Cdk3, Cdk4, Cdk6, Cdk7 and Cdk8) during lens differentiation. Differentiating lens fiber cells were separated from lens epithelial cells by microdissection of developing rat lenses [embryonic day 16 (E16) to postnatal day 8 (P8)] and Cdk expression was assessed by RT-PCR and immunoblotting. Two Cdks (Cdk3 and Cdk6) were not expressed in lens fiber cells or epithelial cells during this developmental period. In the lens epithelium, we detected proteins and mRNAs corresponding to all other Cdks examined (Cdk2, Cdk4, Cdk7, Cdk8) throughout this developmental period. Epithelial cells showed significant Cdk2 activity, which decreased with developmental age, but no significant activity was detected for Cdk4, Cdk7, or Cdk8. Fiber cells contained all four Cdk proteins and the corresponding Cdk mRNAs except for Cdk2 mRNA. None of the Cdks examined showed significant kinase activity in fiber cells. Immunoprecipitates of Cdk2 and Cdk4 from fiber cells contained p57(kip2), supporting the view that this Cdk inhibitor blocks the activity of these Cdks in lens fibers. In contrast, p57(kip2)did not co-immunoprecipitate with Cdk5 from lens fibers. These findings suggest that the differential affinity of p57(kip2)for members of the Cdk family may provide a mechanism for specific regulation of individual Cdks during fiber cell differentiation.
Insights
Lens differentiation involves specific cyclin-dependent kinases (Cdks). The Cdk inhibitor p57(kip2) selectively regulates Cdk activity in fiber cells, crucial for eye development.
Area of Science:
- Cell biology
- Developmental biology
- Molecular biology
Background:
- Lens fiber cell differentiation is a complex process involving precise cell cycle regulation.
- Cyclin-dependent kinases (Cdks) are key regulators of the cell cycle, but their specific roles in lens differentiation are not fully understood.
- Previous studies identified Cdk1 and Cdk5 as active in differentiating lens fiber cells.
Purpose of the Study:
- To investigate the expression and regulation of six additional cyclin-dependent kinases (Cdk2, Cdk3, Cdk4, Cdk6, Cdk7, and Cdk8) during rat lens differentiation.
- To elucidate the role of the Cdk inhibitor p57(kip2) in regulating Cdk activity within lens fiber cells.
Main Methods:
- Microdissection of developing rat lenses (embryonic day 16 to postnatal day 8).
- Assessment of Cdk expression using reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting.
- Kinase activity assays and co-immunoprecipitation studies to analyze Cdk-inhibitor interactions.
Main Results:
- Cdk3 and Cdk6 were not expressed during lens development.
- Lens epithelial cells expressed Cdk2, Cdk4, Cdk7, and Cdk8, with significant Cdk2 activity.
- Lens fiber cells expressed Cdk2, Cdk4, Cdk7, and Cdk8 proteins and mRNAs (except Cdk2 mRNA), but showed no significant kinase activity for these Cdks.
- p57(kip2) co-immunoprecipitated with Cdk2 and Cdk4 in fiber cells, suggesting inhibition, but not with Cdk5.
Conclusions:
- Lens fiber cell differentiation involves the specific expression and regulation of certain cyclin-dependent kinases.
- The Cdk inhibitor p57(kip2) plays a critical role in suppressing Cdk2 and Cdk4 activity in differentiating lens fiber cells.
- Differential binding affinity of p57(kip2) to various Cdks may represent a key mechanism for precise Cdk regulation during lens fiber cell development.